DK145801B - PROCEDURE FOR CULTIVATING A FUNGI RELATED TO THE GENE CORIOLUS - Google Patents

Description

(19) DENMARK

^ (12) PUBLICATION WRITING (11) 145801 B

DIRECTORATE OF THE PATENT 06 BRAND

(21) Application No. 3 ^ 7 ^ / 77 (51) IntCI.1 C 12 N 1/14 (22) Filing Day 3 · Aug. 1977 (24) Race day 3 Aug. 1977 (41) Aim. available Feb 4 1978 (44) Submitted Mar 7 1 9¾ (86) International application # - (86) International filing day (85) Transfer day - (62) Master application no -

(30) Priority 3. aUg. 1976, 51/9307 ^, JP Aug 30 1976, 51/103380, JP

(71) Applicant KUREHA KAGAKU K0GY0 KABUSHIKI KAISHA, Tokyo, JP.

(72) Inventor Chikao Yoshikumi, JP: Toshihiko Wada, JP: Hlromitsu

Makita, JP: KAnzaburo Suzuki, JP: Azuraa Ohkubo, JP: et al.

(74) Full-time engineering firm Lehmann & Ree.

(54) Method for growing a fungus belonging to the genus Coriolus.

The invention relates to a method for growing a fungus belonging to the genus Coriolus, preferably Coriolus versicolor (Fr.) Quél., Coriolus consors (Berk.) Imaz., Coriolus hir-sutus (Fr.) Quél e]. Is Coriolus pargamenus (Fr. .) Pat. under submerse conditions with aeration and stirring and using an aqueous liquid nutrient substrate containing saccharide as carbon source and in addition at least one substance selected from yeast extract,

S

- peptone, casamic acid and meat extract.

Generally, the aqueous liquid substrates of the X 1 type containing at least one substance are selected from yeast extract, peptone, casamino acid and meat extract as nutritional source (nitrogen source) and further contain saccharide (such as glucose, starch, fructose, etc.). ) as a carbon source and one or more inorganic salts (such as phosphate, magnesium salt, etc.) suitable for use in the cultivation of Basidiomyceter, but when a fungus belonging to the genus Coriolus is subjected to submergence using such an aqueous liquid substrate, a vigorous foaming takes place on the surface of the liquid medium during cultivation, and consequently, it is extremely difficult to carry out the cultivation efficiently and uniformly using a large amount of medium in a fermentor.

Therefore, by carrying out submergence of a fungus belonging to the genus Coriolus using an aqueous liquid medium with foaming propensity as mentioned above, it is necessary to reduce the feeding of substrate in the fermentor (to a level of about 40-60% of the capacity of the fermentor). However, as a significant number of days are usually required for growing Basidiomyceter, this reduction in substrate feeding leads to productivity decline, which is a serious economic disadvantage.

Various foaming control devices are available, so a foam attenuator has been mounted on the stirrer in the fermentor, but none of this equipment is capable of suppressing the foaming tendency of the substrate to allow increase substrate feeding. Attempts have also been made to regulate foaming by decreasing the aeration and stirring rates in the substrate, but this approach is also undesirable as it slows the growth rate of the mycelium of the Basidiomycetes. Furthermore, it has been proposed to dilute the liquid substrate in order to suppress the foaming and increase the substrate charge, but this experiment suffers from the disadvantage that the batch is reduced to an unacceptable degree.

Further studies have now been carried out on the foaming phenomenon occurring in the cultivation of fungi of the genus Coriolus using an aqueous liquid substrate as mentioned above, and on the basis of these studies the important recognition that the foaming is powerful at the initial stage of cultivation, it decreases rapidly as the course of cultivation reaches a certain stage, and if the medium or at least one component of the medium is therefore supplemented in appropriate portions at the times when the foaming phenomenon decreases during the cultivation of the Basidiomycetes, the mycelium can be provided. in surprisingly high yield.

The object of the invention is thus to provide a new method for growing mushrooms belonging to the genus Coriolus, by which the foaming medium of the culture medium is controlled so as to ensure a smooth course of the cultivation and to obtain a better utilization of the capacity of the fermentor.

This is achieved by a method according to the invention of the kind referred to in the preamble, which is characterized in that the starting charge of nutrient substrate is adjusted to be less than approx. 70% of the fermentor contents used and that the substrate or at least one of the substrate's nutritional components is added as the foaming in the substrate decreases during the growing period.

The conspicuous feature of the invention is that the aqueous liquid substrate used in the cultivation of the Basiomyiomycetes is not all introduced into the fermenter at one time, as is the case with the conventional process, but initially introduced in an amount of ca. 70% of the capacity of the fermenter used, and that the substrate or at least one component of the nutrient substrate is appropriately supplied at the time when the foaming phenomenon decreases with the further course of cultivation.

In the present invention, the initial scarcity of the substrate, although varying somewhat with the composition of the medium used, the size and shape of the fermentor, the rate of stirring during cultivation, and other factors, are usually in the range of from 40 to 70% of the internal space content of the fermentor used.

The time and amount of each additional charge of the substrate or at least one component of the medium constituting a nutrient source is suitably determined during the culture period by observing the foaming conditions in the substrate through a window provided in the upper part of the fermenters. It is recommended to add an additional amount of medium or at least one component at a time when the phenomenon of foaming has subsided to a certain extent. Such additional feeds of the medium or at least one component may be carried out either batchwise or continuously, subject to the foaming conditions. These supplemental fillings can increase the total feeding of the medium to approx. 85% of the fermenter content.

In the cultivation of mushrooms of the genus Coriolus, great progress is not seen in mycelial growth in the early stages of cultivation, and the amount of nutrients in the substrate used by the Basidiomycetes is limited, so that there is only a slight change in the composition of the medium, while the heavy foaming occurs during the initial phase of cultivation, but it has been found that there is then a sharp decrease in foaming with rapid growth of the Basidiomycetes. Therefore, even if the substrate feeding is regulated to remain below ca. 70% of the fermenter's room contents during the initial stage of cultivation where heavy foaming occurs, the final yield of the mycelium is improved when a sufficient supply of substrate or a component of the substrate is provided by the supplementary batches of the last stage of growth, where the growth of the Basidiomycetes becomes rapid. In case the substrate is used for supplemental charges, it is preferable to set the initial charge so that it is less than approx.

60% of the volume content of the ferments used.

The aqueous liquid substrate used in the present invention contains saccharide such as glucose, lactose, starch, etc. as a carbon source and at least one element of the group consisting of yeast extract, peptone, casamic acid and meat extract as a nutritional source, and this nutrient source is preferably present in an amount of more than 0.1% by weight. Such an aqueous liquid medium, when used for submerging Basidiomyceter, tends to provide strong foaming on the surface of the liquid substrate. The term "substrate component (s)" added to the medium in accordance with the progress of the culture means the nutrient source, i.e. at least one selected from yeast extract, peptone, casamic acid and meat extract.

The fungi used by the genus Coriolus are well known in the art, and their mycological properties are explained in "COLORED ILLUSTRATION OP FUNGI OF JAPAN" by Rokuya Imazeki and Tsuguo Hongo, Volume I, 1974, and Volume II, 1975.

It should also be noted that polysaccharide is formed as a major cultural product in the resulting mycelium of the fungi of the genus Coriolus and in the substrate. This polysaccharide has various pharmacological effects and also many uses as a food additive. Especially when the species referred to above as preferred are used, polysaccharide with very good antitumorigenic effect can be provided.

Such polysaccharide can be separated and recovered by extracting the mycelium obtained by culturing the invention and the fermented substrate with an aqueous solvent.

The invention will now be described in more detail by means of some embodiments.

Example 1; 1000 liters of an aqueous liquid substrate containing 10 wt.% Glucose, 1.5 wt.% Yeast extract, 0.2 wt.% Malt extract, 0.1 wt.% MgSO ^^ 71 ^0 and 0.1 wt.% KE₂ PO₂ were loaded into a fermenter shaped as a vertical container (manufactured by Marubishi Physicochemical Industry Co., Ltd.) having a capacity of 3 2 m and a flat blade turbine stirrer with two levels, and after sterilization in a known manner, the medium was inoculated with 20 liters of slurry of mycelium. Coriolus versicolor (Pr.) Quél., Previously grown as seed culture in a 50 liter glass fermenter, and then immediately commenced cultivation at an aeration rate of 500 l / min. (0.5 liters of air supply per liter of substrate per minute, i.e. 0.5 1/1 / min), a turbine speed of 150 rpm and a cultivation temperature of 26 ° C. Foams formed which rose to the upper part of the fermenter during the initial stage of cultivation, but the foam began to subside after 15-20 hours. Therefore, during the 24 hours from the start of the cultivation, an additional 200 liters of the sterilized substrate of the same composition as above was added over a period of one hour and the culture was continued, with the aeration rate adjusted to 600 l / min. Foams that risen again were observed, but as this subsided over time, an additional 200 liters of substrate was again added under the same conditions as the first supplemental charge in the 48th hour from the start of cultivation, the cultivation being continued further under control of the aeration rate to 700 1 / min. Foam rose for the third time but declined again over time, so that in the 72 hour from the beginning of cultivation 200 liters of medium were added under the same conditions as the first and second supplementary additions and the cultivation was continued further under control of the aeration rate to 800 l / mine. These three additional additions of substrate caused the total amount of substrate added to constitute 1600 liters or 80% of the fermenter's volume content, approximately constituting the limiting amount of feed, so that the cultivation was continued under these conditions and ceased on the 7th day after cultivation. 145801 gene start. At the end of the culture, the mycelium was centrifugally separated from the substrate and dried. The yield was 15.6 g / l.

Example 2: 1000 liters of aqueous liquid substrate containing 15% by weight glucose, 2.25% by weight yeast extract, 0.2% by weight malt extract, 0.1% by weight MgSO 2 -TH 2 O and 0.1% by weight KI 2 PO 2 were loaded into a fermentor in the form of a vertical container with a space content of 2 m and with a flat blade turbine stirrer with 2-storey blades (manufactured by Marubishi Physicochemical Ind.) and after sterilization in a known manner, the substrate was inoculated with 20 liters of slurry of mycelium by Coriolus versicolor ( Fr.) Quél., Previously grown as seed culture in a 50 liter glass fermenter, and then the cultivation started immediately. with an aeration rate of 500 l / min. (0.5 liters of air supply per liter of medium per minute, i.e. 0.5 1/1 / min), a turbine speed of 150 rpm and a cultivation temperature of 26 ° C. Foam rose to the upper part of the fermenter during the first phase of cultivation, but this began to diminish after 30-40 hours, so that in the 48 hours from the start of cultivation 200 liters of sterilized substrate of the same composition as the initially filled substrate and culture continued, with the aeration rate adjusted to 600 l / min. Again, foam increased, but this subsided over time, so that in the 72 hour from the start of cultivation, 200 liters of substrate was added again under the same conditions as the first supplementary charge, and the cultivation was continued further, with the aeration rate adjusted to 700 l / min. . For the third time, foam was seen to rise, but again decreased over time, so that in the 96th hour from the start of cultivation, 200 liters of substrate was added again under the same conditions as for the previous first and second supplements and the aeration rate was adjusted to 800 l / min. These three supplemental additions caused the total amount of substrate supplied to be 1600 liters or 80% of the fermenter's volume content. Since this was close to the limit of the amount of feed that could be fed, cultivation was continued without further substrate supply and ended on the 7th day from the start of cultivation. After completion of the culture, the mycelium was centrifuged from the medium and dried to give a total yield of 18.4 g / l.

For comparison purposes, the experiments below were also carried out.

Comparative Example 1; 1,728,801 1,200 liters of substrate of the same composition as in Example 1 were added at once to a container-shaped fermenter as in Example 1, and after sterilization in a known manner, the substrate was inoculated with 20 liters of slurry of mycelium by Coriolus versicolor (Fr.) Quél. previously grown as seed culture in a 50 liter glass fermenter, after which the cultivation was immediately started at an aeration rate of 500 Ι / min, a turbine speed of 150 rpm and a cultivation temperature of 26 ° C, and cultivation was maintained for 7 days. The yield of mycelium obtained in the same manner as in Example 1 after the end of cultivation was 16.9 g / l.

The results obtained show that the yield of mycelium per rather, the amount of unit substrate is higher in Comparative Example 1 than in Example 1, but in the former it was impossible to supply the substrate in a total amount of more than 60% of the fermenter's volume content due to excessive foaming in the substrate, thereby producing mycelium per unit volume. batch became smaller than in Example 1. The results are shown in Table 1 below.

Comparative Example 2:

The cultivation was carried out according to the same procedure as in Comparative Example 1, except that 1000 liters of substrate with the same composition as in Example 2 were added at one time. In practice, it was impossible to introduce more than 1000 liters of substrate due to excessive foaming in the substrate. The results are shown in Table 1.

Comparative Example 3:

Cultivation was carried out according to the same procedure as in Comparative Example 1 except that at one time 1600 liters of substrate containing 5% by weight glucose, 0.75% by weight yeast extract, 0.2% by weight malt extract, 0.1% by weight MgSO 2 * 7H 2 O and 0 were added. In this case, the amount of substrate feed was equated with the total feed in Examples 1 and 2 by diluting the substrate used. That is, the phenomenon of foaming was reduced by using the diluted medium. The results obtained were as shown in Table 1.

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Example 3: 9 145801 In this example, a substrate component was used as a food source for supplemental application instead of the substrate itself.

35 liters of the liquid substrate containing glucose and yeast extract as shown in Table 2 was added to a 50 liter fermenter in the form of a vertical container and after sterilization in a known manner, the substrate mixture was inoculated with a slurry of mycelium (0.01% measured as mycelium). by Coriolus versicolor (Fr.) Quél. CM-101, deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba, Japan, under the deposit number FERM-P No. 2412 on December 25, 1973, which was a slurry derived from a culture of shaking and cultivation was immediately commenced with an aeration rate of 0.5 l / l / min, a stirrer speed of 250 rpm and a cultivation temperature of 25 + 2 ° C. Foam rose to the upper part of the fermenter during the initial stage of cultivation, but it began to diminish after 15-20 hours, so that 24 hours after the start of the cultivation, 2 liters of an aqueous solution containing yeast extract was added and the cultivation continued. the aeration rate being adjusted to 0.6 l / l / min. Foam rose again but diminished over time, so that 48 hours after the start of cultivation, 2 liters of the aqueous solution was again added and the cultivation continued, setting the aeration rate to 0.6 1/1 / min. Similarly, an additional 2 liters of the aqueous solution was again added once 72 hours after the start of cultivation, and the cultivation was continued further.

These three additional aqueous solution additions plus the initial charge caused the total substrate feed to be 41 liters or 82% of the fermenter's volume content. Since this was roughly the limit of the feed quantity, cultivation was continued with this amount of substrate and ended on the 7th day after the start of cultivation.

After completion of the culture, the mycelium was centrifuged from the substrate and dried. The yield and production were 19.8 g / l and 810 g respectively as shown in Table 2 below.

For comparison purposes, the mycelium was grown by Coriolus versicolor (Fr.) Quél. in the same ferments as that used in Example 3 without further addition of yeast extract to the aqueous liquid substrate mixed with a relatively high amount of yeast extract compared to glucose as shown in Table 2. In this case it was impossible to add more than 25 liters of medium as a result of excessive foaming. The yield of mycelium after 7 days of cultivation was 22.0 g / l and the production was 550 g.

These results show that the process of the invention is capable of substantially increasing mycelium production under conditions where the same ferments are used.

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Example 4:

Mycelium by Coriolus versicolor (Fr.) Quél. CM-101 was grown by the same method and using the same 50 liter ferments as in Example 3. 30 liters of a substrate composed of glucose and yeast extract as shown in Table 3 were initially filled and inoculated with the slurry of Coriolus mycelium. At the 48th and 72nd hours from the start of cultivation, an additional 5 liters of yeast extract was added and the cultivation of the mycelium was continued further. The results are shown in Table 3.

As a comparative example, a similar culture was carried out using a substrate mixed with a high amount of yeast extract relative to glucose and without carrying out any further application of yeast extract to the substrate. In this case, strong foaming occurred, as was the case for the comparative example of Example 3, and this made it impossible to add more than 20 liters of substrate. As a result, mycelium production was as low as given in Table 3 below.

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For further experimental elucidation of the utility of the method of the invention for growing mushrooms of the genus Coriolus, the following experiments have been carried out. In these cultivation experiments, the fungal species Coriolus consors (Berk.) Imaz., Coriolus hirsutus (Fr.) Quél. and Coriolus pargamenus (Fr.)

Pat. used.

By a procedure similar to that described in Example 1, three strains of the respective fungal species Coriolus consors (Berk.) Imaz., Coriolus hirsutus (Fr.) Quél. and Coriolus pargamenus (Fr.) Pat., which is deposited publicly under the respective landfill numbers FERM-P 988, 2711 and 2712. The culture medium was also used as in Example 1.

The results obtained, listed in the table below, included mycelial yield, recovered amount of protein-bound polysaccharides obtained from the respective mycelia by the same procedure as the one used in Example 1, and elemental analytical data for the polysaccharides thus obtained.

Species: Coriolus consors Coriblus hirsutus Coriolus pargamenus

Deposit No .: FERM-P 988 FERM-P 2711 FERM-P 2712

Example: 567

Obtained mycelial amount (g / l) 1.65 1.50 1.60

Extracted amount of protein-bound polysaccharide (g) 3.1 2.7 2.9

Elemental analysis (%)

Carbon 40.1 38.6 39.2

Hydrogen 6.5 6.6 6.2

Nitrogen 2.4 2.1 2.0

The above results show that the method of the invention is not only applicable to the cultivation of the fungus species Coriolus versicolor (Fr.) Quél. but is generally useful for cultivating fungal species belonging to the genus Coriolus.